Updated on 2022/05/17

写真a

 
AKANUMA, Satoshi
 
Affiliation
Faculty of Human Sciences, School of Human Sciences
Job title
Professor

Concurrent Post

  • Faculty of Human Sciences   School of Human Sciences (Online Degree Program)

  • Faculty of Human Sciences   Graduate School of Human Sciences

Degree

  • 東京工業大学   博士(理学)

Research Experience

  • 2020.04
    -
    Now

    Waseda University   Faculty of Human Sciences

  • 2015.04
    -
    2020.03

    Waseda University Faculty of Human Sciences

  • 2007.04
    -
    2015.03

    Tokyo University of Pharmacy and Life Sciences   School of Life Science

  • 2006.04
    -
    2007.03

    Tokyo University of Pharmacy and Life Sciences   School of Life Science

  • 2005.04
    -
    2006.03

    食品総合研究所   特別研究員

  • 2004.10
    -
    2005.03

    Tokyo University of Pharmacy and Life Sciences   School of Life Science

  • 2003.04
    -
    2004.09

    科学技術振興機構   バイオインフォマティクス推進研究員

  • 2001.05
    -
    2003.02

    University of Cologne

  • 1998.04
    -
    2001.04

    理化学研究所   基礎科学特別研究員

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Professional Memberships

  •  
     
     

    THE SOCIETY FOR THE STUDY OF THE ORIGIN AND EVOLUTION OF LIFE-JAPAN

  •  
     
     

    THE JAPANESE BIOCHEMICAL SOCIETY

  •  
     
     

    THE MOLECULAR BIOLOGY SOCIETY OF JAPAN

  •  
     
     

    JAPAN GEOSCIENCE UNION

  •  
     
     

    SOCIETY OF EVOLUTIONARY STUDIES, JAPAN

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    The Society for Biotechnology, Japan

  •  
     
     

    THE BIOPHYSICAL SOCIETY OF JAPAN

  •  
     
     

    PROTEIN SCIENCE SOCIETY OF JAPAN

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Research Areas

  • Biophysics

  • Biofunction and bioprocess engineering

  • Evolutionary biology

Research Interests

  • 祖先型再構成

  • 耐熱性酵素

  • 好熱菌

  • 生命の起源

  • 分子進化

  • 進化分子工学

  • タンパク質工学

▼display all

Papers

  • EXPERIMENTAL TEST OF PLAUSIBLE REDUCED AMINO ACID SETS IN PRIMORDIAL PROTEINS

    Satoshi Akanuma

    Viva Origino   48 ( 3 ) 4  2020.11  [Refereed]  [Invited]

    Authorship:Lead author, Last author, Corresponding author

  • Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties.

    Ryutaro Furukawa, Wakako Toma, Koji Yamazaki, Satoshi Akanuma

    Scientific reports   10 ( 1 ) 15493 - 15493  2020.09  [International journal]

     View Summary

    Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

    DOI PubMed

  • Reconstruction and Characterization of Thermally Stable and Catalytically Active Proteins Comprising an Alphabet of ~ 13 Amino Acids.

    Madoka Kimura, Satoshi Akanuma

    Journal of molecular evolution   88 ( 4 ) 372 - 381  2020.05  [International journal]

     View Summary

    While extant organisms synthesize proteins using approximately 20 kinds of genetically coded amino acids, the earliest protein synthesis system is likely to have been much simpler, utilizing a reduced set of amino acids. However, which types of building blocks were involved in primordial protein synthesis remains unclear. Herein, we reconstructed three convergent sequences of an ancestral nucleoside diphosphate kinase, each comprising a 10 amino acid "alphabet," and found that two of these variants folded into soluble and stable tertiary structures. Therefore, an alphabet consisting of 10 amino acids contains sufficient information for creating stable proteins. Furthermore, re-incorporation of a few more amino acid types into the active site of the 10 amino acid variants improved the catalytic activity, although the specific activity was not as high as that of extant proteins. Collectively, our results provide experimental support for the idea that robust protein scaffolds can be built with a subset of the current 20 amino acids that might have existed abundantly in the prebiotic environment, while the other amino acids, especially those with functional sidechains, evolved to contribute to efficient enzyme catalysis.

    DOI PubMed

  • Establishment of mesophilic-like catalytic properties in a thermophilic enzyme without affecting its thermal stability

    Satoshi Akanuma, Mizumo Bessho, Hikono Kimura, Ryutaro Furukawa, Shin ichi Yokobori, Akihiko Yamagishi

    Scientific Reports   9 ( 1 )  2019.12  [Refereed]

     View Summary

    © 2019, The Author(s). Thermophilic enzymes are generally more thermally stable but are less active at moderate temperatures than are their mesophilic counterparts. Thermophilic enzymes with improved low-temperature activity that retain their high stability would serve as useful tools for industrial processes especially when robust biocatalysts are required. Here we show an effective way to explore amino acid substitutions that enhance the low-temperature catalytic activity of a thermophilic enzyme, based on a pairwise sequence comparison of thermophilic/mesophilic enzymes. One or a combination of amino acid(s) in 3-isopropylmalate dehydrogenase from the extreme thermophile Thermus thermophilus was/were substituted by a residue(s) found in the Escherichia coli enzyme at the same position(s). The best mutant, which contained three amino acid substitutions, showed a 17-fold higher specific activity at 25 °C compared to the original wild-type enzyme while retaining high thermal stability. The kinetic and thermodynamic parameters of the mutant showed similar patterns along the reaction coordinate to those of the mesophilic enzyme. We also analyzed the residues at the substitution sites from a structural and phylogenetic point of view.

    DOI PubMed

  • The Common Ancestor of All Modern Life

    Akanuma Satoshi

    Astrobiology:From the Origins of Life to the Search for Extraterrestrial Intelligence (eds. Yamagishi A, Kakegawa T, Usui T), Springer     91 - 103  2019.03  [Refereed]  [Invited]

    DOI

  • 祖先型再構成タンパク質を用いた原始アミノ酸組成の探索

    赤沼哲史

    生物物理   59 ( 1 ) 23 - 25  2019.01  [Refereed]  [Invited]

    DOI

  • Comprehensive reduction of amino acid set in a protein suggests the importance of prebiotic amino acids for stable proteins

    Rei Shibue, Takahiro Sasamoto, Masami Shimada, Bowen Zhang, Akihiko Yamagishi, Satoshi Akanuma

    Scientific Reports   8 ( 1 ) 1227  2018.12  [Refereed]

     View Summary

    Modern organisms commonly use the same set of 20 genetically coded amino acids for protein synthesis with very few exceptions. However, earlier protein synthesis was plausibly much simpler than modern one and utilized only a limited set of amino acids. Nevertheless, few experimental tests of this issue with arbitrarily chosen amino acid sets had been reported prior to this report. Herein we comprehensively and systematically reduced the size of the amino acid set constituting an ancestral nucleoside kinase that was reconstructed in our previous study. We eventually found that two convergent sequences, each comprised of a 13-amino acid alphabet, folded into soluble, stable and catalytically active structures, even though their stabilities and activities were not as high as those of the parent protein. Notably, many but not all of the reduced-set amino acids coincide with those plausibly abundant in primitive Earth. The inconsistent amino acids appeared to be important for catalytic activity but not for stability. Therefore, our findings suggest that the prebiotically abundant amino acids were used for creating stable protein structures and other amino acids with functional side chains were recruited to achieve efficient catalysis.

    DOI PubMed

  • Reduced amino acid set proteins suggest a role of prebiotic amino acids in primitive protein synthesis

    Shibue R, Akanuma S

    Viva Origino   46   4  2018.09  [Refereed]

    Authorship:Last author, Corresponding author

  • Creation of artificial protein–protein interactions using α-helices as interfaces

    Sota Yagi, Satoshi Akanuma, Akihiko Yamagishi

    Biophysical Reviews   10 ( 2 ) 411 - 420  2018.04  [Refereed]  [Invited]

     View Summary

    Designing novel protein–protein interactions (PPIs) with high affinity is a challenging task. Directed evolution, a combination of randomization of the gene for the protein of interest and selection using a display technique, is one of the most powerful tools for producing a protein binder. However, the selected proteins often bind to the target protein at an undesired surface. More problematically, some selected proteins bind to their targets even though they are unfolded. Current state-of-the-art computational design methods have successfully created novel protein binders. These computational methods have optimized the non-covalent interactions at interfaces and thus produced artificial protein complexes. However, to date there are only a limited number of successful examples of computationally designed de novo PPIs. De novo design of coiled-coil proteins has been extensively performed and, therefore, a large amount of knowledge of the sequence–structure relationship of coiled-coil proteins has been accumulated. Taking advantage of this knowledge, de novo design of inter-helical interactions has been used to produce artificial PPIs. Here, we review recent progress in the in silico design and rational design of de novo PPIs and the use of α-helices as interfaces.

    DOI PubMed

  • Selection of a platinum-binding sequence in a loop of a four-helix bundle protein

    Sota Yagi, Satoshi Akanuma, Asumi Kaji, Hiroya Niiro, Hayato Akiyama, Tatsuya Uchida, Akihiko Yamagishi

    Journal of Bioscience and Bioengineering   125 ( 2 ) 192 - 198  2018.02  [Refereed]

     View Summary

    Protein–metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications.

    DOI PubMed

  • Coarse-grained molecular dynamics simulation of sulerythrin and LARFH for producing protein nanofibers

    Takashi Ozawa, Hironao Yamada, Takeshi Miyakawa, Ryota Morikawa, Sota Yagi, Satoshi Akanuma, Akihiko Yamagishi, Masako Takasu

    ACM International Conference Proceeding Series     43 - 47  2018.01  [Refereed]

     View Summary

    Artificial creation of fibers utilizing proteins has been a target of bionanotechnology. Yagi et al. succeeded in designing artificial protein fibers using two types of proteins: LARFH and sulerythrin. Binding interfaces were designed for sulerythrin and LARFH by introducing mutations, and the fibrous structures were confirmed by atomic force microscopy. However, branching was observed in the fibrous structure, possibly because of non-specific interactions between the proteins. In this study, we analyzed the behavior and binding sites of sulerythrin mutants and LARFH mutants using coarse-grained molecular dynamics (MD) simulation. Binding simulations were performed for a system of one sulerythrin and one LARFH, and also of two sulerythrin molecules and four LARFH molecules. These results suggested that glutamic acids originally possessed by sulerythrin contribute to non-specific binding at sites other than the designed interfaces.

    DOI

  • Activation of macrophages by a laccase-polymerized polyphenol is dependent on phosphorylation of Rac1.

    Katsuya Tajima, Satoshi Akanuma, Akiko Matsumoto-Akanuma, Daisuke Yamanaka, Ken-Ichi Ishibashi, Yoshiyuki Adachi, Naohito Ohno

    Biochemical and biophysical research communications   495 ( 3 ) 2209 - 2213  2018.01  [Refereed]  [International journal]

     View Summary

    Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.

    DOI PubMed

  • 人類への進化のはじまり:生命の起源と全生物の最後の共通祖先

    赤沼哲史, 渋江怜

    人間科学研究   31 ( 2 ) 139 - 145  2018  [Refereed]

  • Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design

    Yuhi Hamuro, Katsuya Tajima, Akiko Matsumoto-Akanuma, Sayaka Sakamoto, Ryutaro Furukawa, Akihiko Yamagishi, Naohito Ohno, Satoshi Akanuma

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   124 ( 6 ) 623 - 629  2017.12  [Refereed]

     View Summary

    Laccases are enzymes that oxidize various aromatic compounds, and therefore they have attracted much attention from the standpoints of medical and industrial applications. We previously isolated the cDNA that codes for a laccase isozyme (Lac2a) from the medicinal mushroom Agaricus brasiliensis (Matsumoto-Akanuma et al., Int. J. Med. Mushrooms, 16, 375-393, 2014). In this study, we first attempted heterologous expression of the wild-type laccase using a Pichia pastoris secretory expression system. However, the trial was unsuccessful most likely because the enzyme was too unstable and degraded immediately after production. Therefore, we improved the stability of the laccase by using a phylogeny-based design method. We created a mutant laccase in which sixteen original residues were replaced with those found in the phylogenetically inferred ancestral sequence. The resulting mutant protein was successfully produced using the P. pastoris secretory expression system and then purified. The designed laccase showed catalytic properties similar to those of other fungal laccases. Moreover, the laccase is highly thermally stable at acidic and neutral pH and is also stable at alkaline pH at moderate temperatures. We expect that the laccase will serve as a useful tool for enzymatic polymerization of di-phenolic compounds. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • Characterization of reconstructed ancestral proteins suggests a change in temperature of the ancient biosphere

    Satoshi Akanuma

    Life   7 ( 3 )  2017.09  [Refereed]  [Invited]

     View Summary

    Understanding the evolution of ancestral life, and especially the ability of some organisms to flourish in the variable environments experienced in Earth’s early biosphere, requires knowledge of the characteristics and the environment of these ancestral organisms. Information about early life and environmental conditions has been obtained from fossil records and geological surveys. Recent advances in phylogenetic analysis, and an increasing number of protein sequences available in public databases, have made it possible to infer ancestral protein sequences possessed by ancient organisms. However, the in silico studies that assess the ancestral base content of ribosomal RNAs, the frequency of each amino acid in ancestral proteins, and estimate the environmental temperatures of ancient organisms, show conflicting results. The characterization of ancestral proteins reconstructed in vitro suggests that ancient organisms had very thermally stable proteins, and therefore were thermophilic or hyperthermophilic. Experimental data supports the idea that only thermophilic ancestors survived the catastrophic increase in temperature of the biosphere that was likely associated with meteorite impacts during the early history of Earth. In addition, by expanding the timescale and including more ancestral proteins for reconstruction, it appears as though the Earth’s surface temperature gradually decreased over time, from Archean to present.

    DOI PubMed

  • Reconstructed ancestral enzymes suggest long-term cooling of Earth's photic zone since the Archean

    Amanda K. Garcia, J. William Schopf, Shin-ichi Yokobori, Satoshi Akanuma, Akihiko Yamagishi

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   114 ( 18 ) 4619 - 4624  2017.05  [Refereed]

     View Summary

    Paleotemperatures inferred from the isotopic compositions (delta O-18 and delta Si-30) of marine cherts suggest that Earth's oceans cooled from 70 +/- 15 degrees C in the Archean to the present similar to 15 degrees C. This interpretation, however, has been subject to question due to uncertainties regarding oceanic isotopic compositions, diagenetic or metamorphic resetting of the isotopic record, and depositional environments. Analyses of the thermostability of reconstructed ancestral enzymes provide an independent method by which to assess the temperature history inferred from the isotopic evidence. Although previous studies have demonstrated extreme thermostability in reconstructed archaeal and bacterial proteins compatible with a hot early Earth, taxa investigated may have inhabited local thermal environments that differed significantly from average surface conditions. We here present thermostability measurements of reconstructed ancestral enzymatically active nucleoside diphosphate kinases (NDKs) derived from light-requiring prokaryotic and eukaryotic phototrophs having widely separated fossil-based divergence ages. The ancestral environmental temperatures thereby determined for these photic-zone organisms-shown in modern taxa to correlate strongly with NDK thermostability-are inferred to reflect ancient surface-environment paleotemperatures. Our results suggest that Earth's surface temperature decreased over geological time from similar to 65-80 degrees C in the Archean, a finding consistent both with previous isotope-based and protein reconstruction-based interpretations. Interdisciplinary studies such as those reported here integrating genomic, geologic, and paleontologic data hold promise for providing new insight into the coevolution of life and environment over Earth history.

    DOI PubMed

  • Mutual positional preference of IPMDH proteins for binding studied by coarse-grained molecular dynamics simulation

    T. Ishioka, H. Yamada, T. Miyakawa, R. Morikawa, S. Akanuma, A. Yamagishi, M. Takasu

    AIP Conference Proceedings   1790 ( 1 ) 020023  2016.12  [Refereed]

     View Summary

    Proteins, which incorporate charged and hydrophobic amino acid residues, are useful as a material of nanotechnology. Among these proteins, IPMDH (3-isopropylmalate dehydrogenase), which has thermal stability, has potential as a material of nanofiber. In this study, we performed coarse-grained molecular dynamics simulation of IPMDH using MARTINI force fields, and we investigated the orientation for the binding of IPMDH. In simulation, we analyzed wild type of IPMDH and the mutated IPMDH proteins, where 13, 20, 27, 332, 335 and 338th amino acid residues are replaced by lysine residues which have positive charge and by glutamic acid residues which have negative charge. Since the binding of mutated IPMDH is advantageous compared with the binding of wild type for one orientation, we suggest that the Coulomb interaction for the binding of IPMDH is important.

    DOI

  • De novo design of protein-protein interactions through modification of inter-molecular helix-helix interface residues

    Sota Yagi, Satoshi Akanuma, Manami Yamagishi, Tatsuya Uchida, Aldhiko Yamagishi

    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS   1864 ( 5 ) 479 - 487  2016.05  [Refereed]

     View Summary

    For de novo design of protein-protein interactions (PPIs), information on the shape and chemical complementarity of their interfaces is generally required. Recent advances in computational PPI design have allowed for de novo design of protein complexes, and several successful examples have been reported. In addition, a simple and easy to-use approach has also been reported that arranges leucines on a solvent-accessible region of an alpha-helix and places charged residues around the leucine patch to induce interactions between the two helical peptides. For this study, we adopted this approach to de novo design a new PPI between the helical bundle proteins sulerythrin and LARFH. A non-polar patch was created on an a-helix of LARFH around which arginine residues were introduced to retain its solubility. The strongest interaction found was for the LARFH variant cysIARFH-IV-3L3R and the sulerythrin mutant 6L6D (K-D = 0.16 mu M). This artificial protein complex is maintained by hydrophobic and ionic interactions formed by the inter-molecular helical bundle structure. Therefore, by the simple and easy to-use approach to create de novo interfaces on the alpha-helices, we successfully generated an artificial PPI. We also created a second LARFH variant with the non-polar patch surrounded by positively charged residues at each end. Upon mixing this LARFH variant with 6L6D, mesh-like fibrous nanostructures were observed by atomic force microscopy. Our method may, therefore, also be applicable to the de novo design of protein nanostructures. (c) 2016 Elsevier B.V. All rights reserved.

    DOI

  • Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp NT3060

    Yasuhisa Fukuda, Asuka Abe, Takashi Tamura, Takahide Kishimoto, Atsushi Sogabe, Satoshi Akanuma, Shin-ichi Yokobori, Akihiko Yamagishi, Katsumi Imada, Kenji Inagaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   121 ( 5 ) 497 - 502  2016.05  [Refereed]

     View Summary

    Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (T-m), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60 degrees C (t(1/2)). Three mutants showed increased stabilities compared to the wild type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • De novo design of protein-protein interactions through modification of inter-molecular helix-helix interface residues.

    Yagi S, Akanuma S, Yamagishi M, Uchida T, Yamagishi A

    Biochimica et biophysica acta   1864 ( 5 ) 479 - 487  2016.05  [Refereed]

    DOI PubMed

  • Chapter 20: Ancestral reconstruction of enzymes

    Akanuma S, Yamagishi A

    Understanding enzymes; Function, Design, Engineering and Analysis (ed. Svendsen A)     683 - 706  2016.04  [Invited]

  • A strategy for designing thermostable enzymes by reconstructing ancestral sequences possessed by ancient life

    Akanuma S, Yamagishi A

    Biotechnology of Extremophiles: Advances and Challenges (ed. Rampelotto PH)     581 - 596  2016.04  [Refereed]  [Invited]

  • ゲノム配列の比較から明らかになった初期生命の好熱性

    赤沼哲史

    地球化学   50 ( 3 ) 199‐210(J‐STAGE) - 210  2016  [Refereed]  [Invited]

    DOI CiNii J-GLOBAL

  • Birth of Archaeal Cells: Molecular Phylogenetic Analyses of G1P Dehydrogenase, G3P Dehydrogenases, and Glycerol Kinase Suggest Derived Features of Archaeal Membranes Having G1P Polar Lipids

    Shin-ichi Yokobori, Yoshiki Nakajima, Satoshi Akanuma, Akihiko Yamagishi

    ARCHAEA-AN INTERNATIONAL MICROBIOLOGICAL JOURNAL   2016   1802675  2016  [Refereed]

     View Summary

    Bacteria and Eukarya have cell membranes with sn-glycerol-3-phosphate (G3P), whereas archaeal membranes contain sn-glycerol-1- phosphate (G1P). Determining the time at which cells with either G3P-lipid membranes or G1P-lipid membranes appeared is important for understanding the early evolution of terrestrial life. To clarify this issue, we reconstructed molecular phylogenetic trees of G1PDH (G1P dehydrogenase; EgsA/AraM) which is responsible for G1P synthesis and G3PDHs (G3P dehydrogenase; GpsA and GlpA/GlpD) and glycerol kinase (GlpK) which is responsible for G3P synthesis. Together with the distribution of these protein-encoding genes among archaeal and bacterial groups, our phylogenetic analyses suggested that GlpA/GlpD in the Commonote (the last universal common ancestor of all extant life with a cellular form, Commonote commonote) acquired EgsA (G1PDH) from the archaeal common ancestor (Commonote archaea) and acquired GpsA and GlpK from a bacterial common ancestor (Commonote bacteria). In our scenario based on this study, the Commonote probably possessed a G3P-lipid membrane synthesized enzymatically, after which the archaeal lineage acquired G1PDH followed by the replacement of a G3P-lipid membrane with a G1P-lipid membrane.

    DOI PubMed

  • Robustness of predictions of extremely thermally stable proteins in ancient organisms

    Satoshi Akanuma, Shin-ichi Yokobori, Yoshiki Nakajima, Mizumo Bessho, Akihiko Yamagishi

    EVOLUTION   69 ( 11 ) 2954 - 2962  2015.11  [Refereed]

     View Summary

    A number of studies have addressed the environmental temperatures experienced by ancient life. Computational studies using a nonhomogeneous evolution model have estimated ancestral G + C contents of ribosomal RNAs and the amino acid compositions of ancestral proteins, generating hypotheses regarding the mesophilic last universal common ancestor. In contrast, our previous study computationally reconstructed ancestral amino acid sequences of nucleoside diphosphate kinases using a homogeneous model and then empirically resurrected the ancestral proteins. The thermal stabilities of these ancestral proteins were equivalent to or greater than those of extant homologous thermophilic proteins, supporting the thermophilic universal ancestor theory. In this study, we reinferred ancestral sequences using a dataset from which hyperthermophilic sequences were excluded. We also reinferred ancestral sequences using a nonhomogeneous evolution model. The newly reconstructed ancestral proteins are still thermally stable, further supporting the hypothesis that the ancient organisms contained thermally stable proteins and therefore that they were thermophilic.

    DOI PubMed

  • Characterization of the low-temperature activity of Sulfolobus tokodaii glucose-1-dehydrogenase mutants

    Taisuke Sugii, Satoshi Akanuma, Sota Yagi, Kazuki Yagyu, Yukiko Shimoda, Akihiko Yamagishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   118 ( 4 ) 367 - 371  2014.10  [Refereed]

     View Summary

    Thermophilic enzymes are potentially useful for industrial processes because they are generally more stable than are mesophilic or psychrophilic enzymes. However, a crucial drawback for their use in such processes is that most thermophilic enzymes are nearly inactive at moderate and low temperatures. We have previously proposed that modulation of the coenzyme-binding pocket of thermophilic dehydrogenases can produce mutated proteins with enhanced low-temperature activities. In the current study, we produced and characterized mutants of an NADP-dependent glucose-1-dehydrogenase from the hyperthermophile Sulfolobus tokodaii in which a predicted coenzyme-binding, non-polar residue was replaced by another non-polar residue. Detailed analyses of the kinetic properties of the wild-type enzyme and its mutants showed that one of the mutants (V254I) had improved kat and k(cat)/K-m values at both 25 degrees C and 80 degrees C. Temperature-induced unfolding experiments showed that the thermal stability of the mutant enzyme was comparable to that of the wild-type enzyme. Calculation of the energetic contribution of the V254I mutation for the dehydrogenase reaction revealed that the mutation destabilizes the enzyme-NADP(+)-glucose ternary complex and reduces the transition-state energy, thus enhancing catalysis. (c) 2014, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

    Masaki Fukuda, Yu Komatsu, Hironao Yamada, Ryota Morikawa, Takeshi Miyakawa, Masako Takasu, Satoshi Akanuma, Akihiko Yamagishi

    MOLECULAR SIMULATION   40 ( 6 ) 498 - 503  2014.05  [Refereed]

     View Summary

    Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein-protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein-protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein-protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein-protein interaction through an intermolecular four-helix bundle formation.

    DOI

  • Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

    Sota Yagi, Satoshi Akanuma, Akihiko Yamagishi

    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS   1844 ( 3 ) 553 - 560  2014.03  [Refereed]

     View Summary

    A non-polar patch on the surface of a protein can cause a reduction in the solubility and stability of the protein, and thereby induce aggregation. However, a non-polar patch may be required so that the protein can bind to another molecule. The mutant 6L-derived from the acidic, dimeric alpha-helical protein sulerythrin and containing six additional leucines arranged to form a non-polar patch on its surface when properly folded-has a substantially reduced solubility in comparison with that of wild-type sulerythrin. This reduced solubility appears to cause 6L to aggregate. To reverse this aggregation, we mutated 6L so that it contained three to six additional glutamates or aspartates that we predicted would surround the non-polar leucine patch on natively folded 6L Although the introduction of three glutamates or aspartates increased solubility, the mutants still aggregate and have a reduced alpha-helical content. Conversely, mutants with six additional glutamates or aspartates appear to exist mostly as dimers and to have the same alpha-helical content as that of wild-type sulerythrin. Notably, the introduction of five lysines or five arginines at the positions held by the glutamates or aspartates did not recover solubility as effectively as did the negatively charged residues. These results demonstrate that negatively charged residues, but not positively charged ones, surrounding a non-polar patch on an acidic protein can completely reverse the decrease in its solubility caused by the patch of non-polar surface residues. (c) 2013 Elsevier B.V. All rights reserved.

    DOI

  • Computer Simulation Analysis of the Protein Binding Interfaces that Form a 4-Helix Bundle Motif

    Masaki Fukuda, Hironao Yamada, Ryota Morikawa, Takeshi Miyakawa, Masako Takasu, Satoshi Akanuma, Akihiko Yamagishi

    JPS Conference Proceedings   1   016020  2014.03  [Refereed]

     View Summary

    The objective of this study is to design the binding interface between proteins that form protein–protein complex. We chose a 4-helix bundle motif as a scaffold for forming the intermolecular binding. Based on this motif, we designed several binding interfaces including some charged and hydrophobic amino acid residues. Molecular dynamics (MD) simulations were performed to explore appropriate amino acid arrangements for the binding interface. We found binding interfaces that may be suitable for inducing the intermolecular binding.

    DOI

  • Coarse-Grained Molecular Dynamics Simulation of IPMDH Proteins

    Yosuke Hirata, Yu Komatsu, Masaki Fukuda, Hironao Yamada, Takeshi Miyakawa, Ryota Morikawa, Satoshi Akanuma, Akihiko Yamagishi, Masako Takasu

    JPS Conference Proceedings   1   016010  2014.03  [Refereed]

     View Summary

    We have performed molecular dynamics simulation of coarse-grained model of IPMDH protein to study the effect of mutation to the binding sites of protein. We replace original amino acids on the surface by negatively or positively charged amino acids. We also study the effect of solvent to the binding of proteins. Our computational study may be useful for future experimental study of protein bindings.

    DOI

  • Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch.

    Yagi S, Akanuma S, Yamagishi A

    Biochimica et biophysica acta   1844 ( 3 ) 553 - 560  2014.03  [Refereed]

    DOI PubMed

  • N-terminal Hydrophobic Amino Acids of Activating Transcription Factor 5 (ATF5) Protein Confer Interleukin 1 beta (IL-1 beta)-induced Stabilization

    Takanori Abe, Masaki Kojima, Satoshi Akanuma, Hiromi Iwashita, Takashi Yamazaki, Ryuichi Okuyama, Kenji Ichikawa, Mariko Umemura, Haruo Nakano, Shigeru Takahashi, Yuji Takahashi

    JOURNAL OF BIOLOGICAL CHEMISTRY   289 ( 7 ) 3888 - 3900  2014.02  [Refereed]

     View Summary

    Activating transcription factor 5 (ATF5) is a stress-response transcription factor that responds to amino acid limitation and exposure to cadmium chloride (CdCl2) and sodium arsenite (NaAsO2). The N-terminal amino acids contribute to the destabilization of the ATF5 protein in steady-state conditions and serve as a stabilization domain in the stress response after CdCl2 or NaAsO2 exposure. In this study, we show that interleukin 1 (IL-1), a proinflammatory cytokine, increases the expression of ATF5 protein in HepG2 hepatoma cells in part by stabilizing the ATF5 protein. The N-terminal domain rich in hydrophobic amino acids that is predicted to form a hydrophobic network was responsible for destabilization in steady-state conditions and served as an IL-1 response domain. Furthermore, IL-1 increased the translational efficiency of ATF5 mRNA via the 5 UTR and phosphorylation of the eukaryotic translation initiation factor 2 (eIF2). ATF5 knockdown in HepG2 cells up-regulated the IL-1-induced expression of the serum amyloid A 1 (SAA1) and SAA2 genes. Our results show that the N-terminal hydrophobic amino acids play an important role in the regulation of ATF5 protein expression in IL-1-mediated immune response and that ATF5 is a negative regulator for IL-1-induced expression of SAA1 and SAA2 in HepG2 cells.

    DOI PubMed

  • Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

    Hirokazu Takahashi, Hiroyuki Yamazaki, Satoshi Akanuma, Hiroko Kanahara, Toshiyuki Saito, Tomoyuki Chimuro, Takayoshi Kobayashi, Toshio Ohtani, Kimiko Yamamoto, Shigeru Sugiyama, Toshiro Kobori

    PLOS ONE   9 ( 2 ) e82624  2014.02  [Refereed]

     View Summary

    We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

    DOI PubMed

  • 全生物の共通祖先遺伝子の復元

    赤沼哲史, 山岸明彦

    バイオサイエンスとインダストリー   72 ( 1 ) 19 - 23  2014.01  [Invited]

    CiNii J-GLOBAL

  • Cloning and Characterization of Laccase DNA from the Royal Sun Medicinal Mushroom, Agaricus brasiliensis (Higher Basidiomycetes)

    Akiko Matsumoto-Akanuma, Satoshi Akanuma, Masuro Motoi, Akihiko Yamagishi, Naohito Ohno

    INTERNATIONAL JOURNAL OF MEDICINAL MUSHROOMS   16 ( 4 ) 375 - 393  2014  [Refereed]

     View Summary

    Laccase isozymes have been identified in several fungi. We report the cloning of 4 laccase genes from the medicinal mushroom Agaricus brasiliensis. The lac1 gene contained a 1560-base pair (bp) open reading frame (ORF) encoding 520 amino acids that was interrupted with 14 introns in genomic DNA. The deduced amino acid sequence indicated a multicopper oxidase signature 1 and 2 multicopper oxidase signature 2. The lac2 gene contained a 1566-bp ORF encoding 522 amino acids that was interrupted with 13 introns in genomic DNA. A number of different nucleotides were observed in whole regions containing the substitution of amino acid residues (lac2a and lac2b). The partial DNA fragments of lac3 and lac4 genes were subcloned using the semi-random two-step polymerase chain reaction method. The lac3 and lac4 genes contained coding sequences with a 1575-bp ORF encoding 525 amino acids and a 1584-bp ORF encoding 528 amino acids, respectively. However, the whole complementary DNA fragment of both laccases could not be amplified with polymerase chain reaction against the complementary DNA library; therefore, introns were deduced based on the GT-AG rule and multiple alignment of laccases from other fungi, which showed high identity. All laccases from A. brasiliensis conserved the fungal laccase signature sequence and suggest 2 subfamilies according to the location of introns and phylogenetic analysis. The genes lac2 and lac4 had a high degree of identity, and the lac2a gene was located upstream of the lac4 gene.

    DOI PubMed

  • Experimental evidence for the thermophilicity of ancestral life

    Satoshi Akanuma, Yoshiki Nakajima, Shin-ichi Yokobori, Mitsuo Kimura, Naoki Nemoto, Tomoko Mase, Ken-ichi Miyazono, Masaru Tanokura, Akihiko Yamagishi

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   110 ( 27 ) 11067 - 11072  2013.07  [Refereed]

     View Summary

    Theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. Here we provide experimental support for the existence of a thermophilic universal common ancestor. We present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (NDK), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of Archaea and of Bacteria. These enzymes display extreme thermal stabilities, suggesting thermophilic ancestries for Archaea and Bacteria. The results are robust to the uncertainties associated with the sequence predictions and to the tree topologies used to infer the ancestral sequences. Moreover, mutagenesis experiments suggest that the universal ancestor also possessed a very thermostable NDK. Because, as we show, the stability of an NDK is directly related to the environmental temperature of its host organism, our results indicate that the last common ancestor of extant life was a thermophile that flourished at a very high temperature.

    DOI PubMed J-GLOBAL

  • Exploring Sequences for Thermally Stable Proteins by the Ancestral Design Method

    AKANUMA Satoshi

    Seibutsu Butsuri   53 ( 3 ) 128 - 133  2013.05  [Refereed]  [Invited]

    DOI CiNii J-GLOBAL

  • 3P090 Creation of a platinum-binding loop on an artificial four-helix bundle protein(01F. Protein: Engineering,Poster)

    Niiro Hiroya, Akanuma Satoshi, Yamagishi Akihiko, Akiyama Yuuto, Uchida Tatuya

    Seibutsu Butsuri   53 ( 1 ) S226  2013

    DOI CiNii

  • 1P023 Evaluation of the designed protein binding interfaces as studied by MD simulation(01A. Protein:Structure,Poster)

    Fukuda Masaki, Yamada Hironao, Miyakawa Takeshi, Morikawa Ryota, Takasu Masako, Akanuma Satoshi, Yamagishi Akihiko

    Seibutsu Butsuri   53 ( 1 ) S109  2013

    DOI CiNii

  • The simulation study of protein-protein interfaces based on the 4-helix bundle structure

    Masaki Fukuda, Yu Komatsu, Ryota Morikawa, Takeshi Miyakawa, Masako Takasu, Satoshi Akanuma, Akihiko Yamagishi

    AIP Conference Proceedings   1518   606 - 609  2013  [Refereed]

     View Summary

    Docking of two protein molecules is induced by intermolecular interactions. Our purposes in this study are: designing binding interfaces on the two proteins, which specifically interact to each other
    and inducing intermolecular interactions between the two proteins by mixing them. A 4-helix bundle structure was chosen as a scaffold on which binding interfaces were created. Based on this scaffold, we designed binding interfaces involving charged and nonpolar amino acid residues. We performed molecular dynamics (MD) simulation to identify suitable amino acid residues for the interfaces. We chose YciF protein as the scaffold for the protein-protein docking simulation. We observed the structure of two YciF protein molecules (I and II), and we calculated the distance between centroids (center of gravity) of the interfaces' surface planes of the molecules I and II. We found that the docking of the two protein molecules can be controlled by the number of hydrophobic and charged amino acid residues involved in the interfaces. Existence of six hydrophobic and five charged amino acid residues within an interface were most suitable for the protein-protein docking. © 2013 American Institute of Physics.

    DOI

  • Comparative genomics of thermophilic bacteria and archaea

    Satoshi Akanuma, Shin Ichi Yokobori, Akihiko Yamagishi

    Thermophilic Microbes in Environmental and Industrial Biotechnology: Biotechnology of Thermophiles     331 - 349  2013.01  [Refereed]  [Invited]

     View Summary

    © Springer Science+Business Media Dordrecht 2013. Elucidation of the origin and the early evolution of life is fundamental to our understanding of ancient living systems and of the ancient global environment where early life evolved. A number of molecular phylogenetic trees have been constructed by comparing the homologous gene sequences. In this chapter, we have reviewed the universal trees constructed based on different types of genetic information. The tree topology was different depending on the type of the gene analyzed as well as the method used. The root of the universal tree is most likely placed between the bacterial branch and the common ancestor of Archaea and Eucarya. However, there are possibilities that the root may be within the bacterial branches. Monophyly of Archaea is rather controversial. Though the rRNA tree suggested the monophyly, other types of the tree are also reported. The conclusive result where the Eucarya originated within/outside of the branch of Archaea is yet to come. The growth temperature of the ancient organism has long been a topic that has interested many scientists. Theoretical works suggested mesophilic, thermophilic, and hyperthermophilic origin of life, depending on the report. Experimental test analyzing the effect of each or combination of ancestral amino acid residues suggested the hyperthermophilic origin of life. However, we cannot totally deny the possible artifact based on the method used for the estimation of ancestral sequences possessed by the ancestral organisms.

    DOI

  • Constructing protein nano-fiber and estimation of the electronic state around metal ions

    Yu Komatsu, Masaki Fukuda, Hironao Yamada, Shuhei Kawamoto, Takeshi Miyakawa, Ryota Morikawa, Masako Takasu, Satoshi Yokojima, Satoshi Akanuma, Akihiko Yamagishi

    INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY   112 ( 24 ) 3750 - 3755  2012.12  [Refereed]

     View Summary

    We aim to construct a nano-fiber using proteins by mixing two kinds of proteins. The binding sites are expected to be formed between two a-helices of one protein and two a-helices of another protein. As model proteins, we use Lac repressor four helix protein (LARFH), sulerythrin, and 3-isopropylmalate dehydrogenase. With these proteins, we performed molecular dynamics (MD) simulations with a coarse-grained (CG) model. In MD simulations with the CG model for LARFH, we found that the molecules approached faster in the system where the mutant protein with positively charged amino acids was placed with the mutant protein with negatively charged amino acids than in the system containing two wild-type protein molecules. In the system with the variants, the number of contact interfaces with the positive variant-negative variant combination after 150 ns simulation is larger than that with the positivepositive variants combination or the negativenegative variants combination. Sulerythrin has two pairs of Fe2+ and Zn2+. Electronic structure calculation was performed around metal ions in sulerythrin. By electronic structure calculation for sulerythrin around metal ions, the electric charge and spin density were estimated. (C) 2012 Wiley Periodicals, Inc.

    DOI

  • 1PT135 Simulation study for designing protein-protein interaction interfaces based on a four-helix bundle protein(The 50th Annual Meeting of the Biophysical Society of Japan)

    Fukuda Masaki, Komatsu Yu, Morikawa Ryota, Miyakawa Takeshi, Takasu Masako, Akanuma Satoshi, Yanagishi Akihiko

    Seibutsu Butsuri   52   S91  2012

    DOI CiNii

  • 2PT203 Experimental approach to the amino acid usage of pre-last universal ancestor(The 50th Annual Meeting of the Biophysical Society of Japan)

    Shimada Masami, Akanuma Satoshi, Shinozaki Kozue, Nakajima Yoshiki, Yamagishi Akihiko

    Seibutsu Butsuri   52   S139  2012

    DOI CiNii

  • Designing the binding surface of proteins to construct nano-fibers

    Komatsu Y, Yamada H, Kawamoto S, Fukuda M, Miyakawa T, Morikawa R, Takasu M, Akanuma S, Yamagishi A

    Prog. Theor. Chem. Phys.   26   555 - 567  2012  [Refereed]

  • Substitutions of Coenzyme-Binding, Nonpolar Residues Improve the Low-Temperature Activity of Thermophilic Dehydrogenases

    Sayaka Hayashi, Satoshi Akanuma, Wakana Onuki, Chihiro Tokunaga, Akihiko Yamagishi

    BIOCHEMISTRY   50 ( 40 ) 8583 - 8593  2011.11  [Refereed]

     View Summary

    Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 -> Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD(+), suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.

    DOI PubMed J-GLOBAL

  • Phylogeny-Based Design of a B-Subunit of DNA Gyrase and Its ATPase Domain Using a Small Set of Homologous Amino Acid Sequences

    Satoshi Akanuma, Shoko Iwami, Tamaki Yokoi, Nana Nakamura, Hideaki Watanabe, Shin-ichi Yokobori, Akihiko Yamagishi

    JOURNAL OF MOLECULAR BIOLOGY   412 ( 2 ) 212 - 225  2011.09  [Refereed]

     View Summary

    We have developed a phylogeny-based design method that has been used to produce mutated proteins with enhanced thermal stabilities. We previously validated the predictive worth of the method by producing and characterizing mutants in which one original residue or a small number of the original residues had been replaced with the one or the ones found in the phylogenetically predicted "ancestral" sequence. For the current study, this method was used to design a sequence for the deepest nodal position of a phylogenic tree composed of 16 gyrase B-subunit sequences, which was then synthesized and characterized. The sequence was inferred from the sequences of 16 extant DNA gyrases and 3 extant type VI DNA topoisomerases. Genes encoding the inferred sequence and its N-terminal ATPase domain were PCR constructed and expressed in Escherichia coli. The full-length designed protein is slightly less thermally stable than is subunit B from the extant thermophilic Thermus thermophilus DNA gyrase, whereas the thermal stability of the designed ATPase domain is more similar to that of the T. thermophilus ATPase domain. Moreover, the designed ATPase domain has significant catalytic activity. Therefore, even a small set of homologous amino acid sequences contains sufficient information to design a thermally stable and functional protein. Because the isolated designed ATPase domain is more thermally stable and catalytically active than is the sequence containing the most frequently occurring amino acids among the 16 gyrases, the phylogenetic approach was superior (in this case, at least) to the consensus approach when the same data set was used to predict the two sequences. (C) 2011 Elsevier Ltd. All rights reserved.

    DOI PubMed J-GLOBAL

  • Roles for the two N-terminal (beta/alpha) modules in the folding of a (beta/alpha)(8)-barrel protein as studied by fragmentation analysis

    Satoshi Akanuma, Akihiko Yamagishi

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   79 ( 1 ) 221 - 231  2011.01  [Refereed]

     View Summary

    The (beta/alpha)(8)-barrel is one of the most abundant folds found in enzymes. To identify the independent folding units and the segment(s) that correspond to a minimum core structure within a (beta/alpha)(8)-barrel protein, fragmentation experiments were performed with Escherichia coli phosphoribosylanthranilate isomerase, which has a single (beta/alpha)(8)-barrel domain. Our previous studies indicated that the central four beta/alpha segments comprise an independent folding unit; whereas, the role(s) of the first two beta/alpha segments in folding had not been clarified prior to this report. Herein, we report the design and synthesis of a series of N-terminally deleted fragments starting with (beta/alpha)(1-5)beta(6) as the parent construct. Analytical gel filtration and urea-induced equilibrium unfolding experiments indicated that deletions within the N-terminal region, that is, within the first two beta/alpha modules, resulted in reduced stability or aggregation of the remaining segments. The (beta/alpha)(3-5)beta(6) segment appeared to fold into a stable structure and deletion of beta(6) from (beta/alpha)(3-5)beta(6) yielded (beta/alpha)(3-5), which did not form native-like secondary structures. However, urea-induced unfolding of (beta/alpha)(3-5), monitored by reduction of tryptophan fluorescence, indicated that the fragment contained a loosely packed hydrophobic core. Taken together, the results of our previous and present fragmentation experiments suggest the importance of the central (beta/alpha)(3-4)beta(5) module in folding, which is a finding that is compatible with our simulated unfolding study performed previously. Proteins 2011; 79: 221-231. (C) 2010 Wiley-Liss, Inc.

    DOI PubMed J-GLOBAL

  • Partial Purification and Characterization of Polyphenoloxidase from Culinary-Medicinal Royal Sun Mushroom (the Himematsutake), Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae)

    Akiko Matsumoto-Akanuma, Satoshi Akanuma, Masuro Motoi, Akihiko Yamagishi, Naohito Ohno

    INTERNATIONAL JOURNAL OF MEDICINAL MUSHROOMS   13 ( 1 ) 73 - 82  2011  [Refereed]

     View Summary

    The Royal Sun mushroom, the Himematsutake culinary-medicinal mushroom, Agaricus brasiliensis has several polyphenoloxidase activities in a broad sense. Here we report the partial purification of tyrosinase-type polyphenoloxidase (PPO). PPO is purified from A. brasiliensis without browning using a two-phase partitioning with Triton X-114 and ammonium sulfate fractionation. Partially denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) staining with L-3,4-dihydroxyphenylalanine was performed and the indicated molecular sizes were approximately 70 kDa and 45 kDa. The purified enzyme is in its latent state and can be activated maximally in the presence of 1.6 mM sodium dodecyl sulfate (SDS). This enzyme catalyzes two distinct reactions, monophenolase and diphenolase activity, and the monophenolase activity showed a lag time typical of polyphenoloxidase. The K(m) value for 4-tert-butylcatechol was quite similar in the presence and absence of SDS, but the apparent V(max) value was increased 2.0-fold by SDS. Mimosine was a typical competitive inhibitor with K(i) values of 138.2 mu M and 281.0 mu M n the presence and absence of SDS, respectively.

    DOI PubMed

  • Mimicking the evolution of a thermally stable monomeric four-helix bundle by fusion of four identical single-helix peptides

    Satoshi Akanuma, Taku Matsuba, Emi Ueno, Naoki Umeda, Akihiko Yamagishi

    JOURNAL OF BIOCHEMISTRY   147 ( 3 ) 371 - 379  2010.03  [Refereed]

     View Summary

    Internal symmetry is a common feature of the tertiary structures of proteins and protein domains. Probably, because the genes of homo-oligomeric proteins duplicated and fused, their evolutionary descendants are proteins with internal symmetry. To identify any advantages that cause monomeric proteins with internal symmetry to be selected evolutionarily, we characterized some of the physical properties of a recombinant protein with a sequence consisting of two tandemly fused copies of the Escherichia coli Lac repressor C-terminal alpha-helix. This polypeptide exists in solution mainly as dimer that likely maintains a four-helix bundle motif. Thermal unfolding experiments demonstrate that the protein is considerably more stable at elevated temperatures than is a homotetramer consisting of four non-covalently associated copies of a 21-residue polypeptide similar in sequence to that of the Lac repressor C-terminal alpha-helix. A tandem duplication of our helix-loop-helix polypeptide yields an even more thermally stable protein. Our results exemplify the concept that fusion of non-covalently assembled polypeptide chains leads to enhanced protein stability. Herein, we discuss how our work relates to the evolutionary selective-advantages realized when symmetrical homo-oligomers evolve into monomers. Moreover, our thermally stable single-chain four-helix bundle protein may provide a robust scaffold for development of new biomaterials.

    DOI PubMed J-GLOBAL

  • Why are thermophilic proteins thermally stable?

    Satoshi Akanuma, Akihiko Yamagishi

    Seikagaku   81 ( 12 ) 1064 - 1071  2009  [Refereed]

    PubMed

  • Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

    Michika Sasaki, Mayumi Uno, Satoshi Akanuma, Akihiko Yamagishi

    PROTEIN ENGINEERING DESIGN & SELECTION   21 ( 12 ) 721 - 727  2008.12  [Refereed]

     View Summary

    In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.

    DOI PubMed J-GLOBAL

  • Experimental evidence for the existence of a stable half-barrel subdomain in the (beta/alpha)(8)-barrel fold

    Satoshi Akanuma, Akihiko Yamagishi

    JOURNAL OF MOLECULAR BIOLOGY   382 ( 2 ) 458 - 466  2008.10  [Refereed]

     View Summary

    The (beta/alpha)(8)-barrel is one of the most common folds functioning as enzymes. The emergence of two (beta/alpha)(8)-barrel enzymes involved in histidine biossynthesis, each of which has a twofold symmetric structure, has been proposed to be a consequence of tandem duplication and fusion of a (beta/alpha)(4)-half-barrel. However, little evidence has been found for the existence of an ancestral half-barrel in the evolution of other (beta/alpha)(8)-barrel proteins. In order to detect remnants of an ancestral half-barrel in the (beta/alpha)(8)-barrel structure of Escherichia coli N-(5'-phosphoribosyl)anthranilate isomerase, we engineered three potential half-barrel units (beta/alpha)(1-4), (beta/alpha)(3-6), and (beta/alpha)(5-8). Among these three arrangements, only (beta/alpha)(3-6) is stable; it exists in equilibrium between monomeric and dimeric forms. Thus, the central segment of N-(5'-phosphoribosyl)anthranilate isomerase from E. coli can serve as a half-barrel precursor. A tandem duplication of (beta/alpha)(3-6) yielded predominantly monomeric structures that were quite stable. This result exemplified that the structural characteristics of moncovalently assembled half-barrels could be improved by duplication and fusion. Moreover, our results may provide information regarding the local structural units that encompass interactions important for the early folding events of this ubiquitous protein conformation. (C) 2008 Elsevier Ltd. All rights reserved.

    DOI PubMed J-GLOBAL

  • Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

    Jun'ichi Wakayama, Hiroshi Sekiguchi, Satoshi Akanuma, Toshio Ohtani, Shigeru Sugiyama

    ANALYTICAL BIOCHEMISTRY   380 ( 1 ) 51 - 58  2008.09  [Refereed]

     View Summary

    We developed a method to measure the. rupture forces between antibody and antigen by atomic force microscopy (AFM), Previous studies have reported that in the measurement of antibody-antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM. (c) 2008 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Identification and characterization of key substructures involved in the early folding events of a (beta/alpha)(8)-barrel protein as studied by experimental and computational methods

    S Akanuma, A Yamagishi

    JOURNAL OF MOLECULAR BIOLOGY   353 ( 5 ) 1161 - 1170  2005.11  [Refereed]

     View Summary

    A number of studies have examined the structural properties of late folding intermediates Of beta/U=alpha)(8)-barrel proteins involved in tryptophan biosynthesis, whereas there is little information available about the early folding events of these proteins. To identify the contiguous polypeptide segments important to the folding of the (beta/alpha)(8)-barrel protein Escherichia coli N-(5'-phosphoribosyl)anthranilate isomerase, we structurally characterized fragments and circularly permuted forms of the protein. We also simulated thermal unfolding of the protein using molecular dynamics. Our fragmentation experiments demonstrate that the isolated (beta/alpha()1-4)beta(5) fragment is almost as stable as the full-length protein. The far and near-UV CD spectra of this fragment are indicative of native-like secondary and tertiary structures. Structural analysis of the circularly permutated proteins shows that if the protein is cleaved within the two N-terminal beta alpha modules, the amount of secondary structure is unaffected, whereas, when cleaved within the central (beta/alpha)(3-4)beta(5) segment, the protein simply cannot fold. An ensemble of the denatured structures produced by thermal unfolding simulations contains a persistent local structure comprised Of beta(3), beta(4) and beta(5). The presence of this three-stranded P-barrel suggests that it may be an important early-stage folding intermediate. Interactions found in (beta/alpha)(3-4)beta(5) may be essential for the early events of ePRAI folding if they provide a nucleation site that directs folding. (c) 2005 Elsevier Ltd. All rights reserved.

    DOI PubMed

  • A detailed unfolding pathway of a (beta/alpha)(8)-barrel protein as studied by molecular dynamics simulations

    S Akanuma, H Miyagawa, K Kitamura, A Yamagishi

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   58 ( 3 ) 538 - 546  2005.02  [Refereed]

     View Summary

    The (P/alpha)(8)-barrel is the most common protein fold. Similar structural properties for folding intermediates of (beta/alpha)(8)-barrel proteins involved in tryptophan biosynthesis have been reported in a number of experimental studies; these intermediates have the last two P-strands and three a-helices partially unfolded, with other regions of the polypeptide chain native-like in conformation. To investigate the detailed folding/unfolding pathways of these (beta/alpha)(8)-barrel proteins, temperature-induced unfolding simulations of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia colt were carried out using a special-purpose parallel computer system. Unfolding simulations at five different temperatures showed a sequential unfolding pathway comprised of several events. Early events in unfolding involved disruption of the last two strands and three helices, producing an intermediate ensemble similar to those detected in experimental studies. Then, denaturation of the first two Pot units and separation of the sixth strand from the fifth took place independently. The remaining central betaalphabetaalphabeta module persisted the longest during all simulations, suggesting an important role for this module as the incipient folding scaffold. Our simulations also predicted the presence of a nucleation site, onto which several hydrophobic residues condensed forming the foundation for the central betaalphabetaalphabeta module.
    (C) 2004 Wiley-Liss, Inc.

    DOI PubMed

  • Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set

    S Akanuma, T Kigawa, S Yokoyama

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   99 ( 21 ) 13549 - 13553  2002.10  [Refereed]

     View Summary

    We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution.

    DOI PubMed

  • Experimental Evidence for Information Redundancy in Amino Acid Sequences: Implication for Protein Folding and Evolution.

    赤沼哲史, 黒田裕

    生物物理   41 ( 5 ) 224 - 229  2001.10

     View Summary

    A protein three-dimensional structure is essentially determined by its amino acid sequence, and thus by the information contained therein. Although "protein folders" have long studied what physical forces are important to stabilize a protein structure, it is only recently that one has experimentally asked the question: how much and what information is prerequisite for stabilizing a protein structure. Here, we describe two recent experiments in which the information contents of two protein sequences have been dramatically decreased and nevertheless the proteins remain active and natively structured. We discuss the implication of these findings both from a protein structure and a molecular evolution point of view.

    DOI CiNii J-GLOBAL

  • Design, X-ray crystallography, molecular modelling and thermal stability studies of mutant enzymes at site 172 of 3-isopropylmalate dehydrogenase from Thermus thermophilus

    CX Qu, S Akanuma, N Tanaka, H Moriyama, T Ohsima

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   57 ( Pt 2 ) 225 - 232  2001.02  [Refereed]

     View Summary

    The relationship between the structure and the thermostability of the 3-isopropylmalate dehydrogenase from Thermus thermophilus was studied by site-directed mutation of a single Ala residue located at the domain interface. The crystal structures of three mutant enzymes, replacing Ala172 with Gly, Val and Phe, were successfully determined at 2.3, 2.2 and 2.5 Angstrom resolution, respectively. Substitution of Ala172 by relatively 'short' residues (Gly, Val or Ile) enlarges or narrows the cavity in the vicinity of the C-beta atom of Ala172 and the thermostability of the enzyme shows a good correlation with the hydrophobicity of the substituted residues. Substitution of Ala172 by the 'longer' residues Leu or Phe causes a rearrangement of the domain structure, which leads to a higher thermostability of the enzymes than that expected from the hydrophobicity of the substituted residues. Mutation of Ala172 to negatively charged residues gave an unexpected result: the melting temperature of the Asp mutant enzyme was reduced by 2.7 K while that of the Glu mutant increased by 1.8 K. Molecular-modelling studies indicated that the glutamate side chain was sufficiently long that it did not act as a buried charge as did the aspartate, but instead protruded to the outside of the hydrophobic cavity and contributed to the stability of the enzyme by enhancing the packing of the local side chains and forming an extra salt bridge with the side chain of Lys175.

    DOI PubMed

  • Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships

    Z Nurachman, S Akanuma, T Sato, T Oshima, N Tanaka

    PROTEIN ENGINEERING   13 ( 4 ) 253 - 258  2000.04  [Refereed]

     View Summary

    Thermal stability of the Thermus thermophilus isopropylmalate dehydrogenase enzyme was substantially lost upon the deletion of three residues from the C-terminus. However, the stability was partly recovered by the addition of two, four and seven amino acid residues (called HD177, HD708 and HD711, respectively) to the C-terminal region of the truncated enzyme. Three structures of these mutant enzymes were determined by an X-ray diffraction method, All protein crystals belong to space group P2(1) and their structures were solved by a standard molecular replacement method where the original dimer structure of the A172L mutant was used as a search model. Thermal stability of these mutant enzymes is discussed based on the 3D structure with special attention to the width of the active-site groove and the minor groove, distortion of beta-sheet pillar structure and size of cavity in the domain-domain interface around the C-terminus. Our previous studies revealed that the thermal stability of isopropylmalate dehydrogenase increases when the active-site cleft is closed (the closed form). In the present study it is shown that the active-site cleft can be regulated by open-close movement of the minor groove located at the opposite side to the active-site groove on the same subunit, through a paperclip-like motion.

    PubMed

  • Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis

    S Akanuma, A Yamagishi, N Tanaka, T Oshima

    EUROPEAN JOURNAL OF BIOCHEMISTRY   260 ( 2 ) 499 - 504  1999.03  [Refereed]

     View Summary

    A thermostabilized mutant of Bacillus subtilis 3-isopropylmalate dehydrogenase (IPMDH) obtained in a previous study contained a set of triple amino acid substitutions. To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizing substitutions, Thr22--> Lys and Met256-->Val, that separately endowed the protein with further stability. We introduced the two mutations into a single enzyme molecule, thus constructing a mutant with overall quintuple mutations. Other studies have suggested that an improved hydrophobic subunit interaction and a rigid type II beta-turn play important roles in enhancing the protein stability. Based on those observations, we successively introduced amino acid substitutions into the mutant with the quintuple mutations by site-directed mutagenesis: Glu253 at the subunit interface was replaced by Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were replaced by Pro, Gly and Glu, respectively, to make the turn more rigid. The thermal stability of the mutants was determined based on remaining activity after heat treatment and first-order rate constant of thermal unfolding, which showed gradual increases in thermal stability as more mutations were included.

    DOI PubMed

  • Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution

    S Akanuma, A Yamagishi, N Tanaka, T Oshima

    PROTEIN SCIENCE   7 ( 3 ) 698 - 705  1998.03  [Refereed]

     View Summary

    We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.

    PubMed

  • Effect of polar side chains at position 172 on thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus

    S Akanuma, CX Qu, A Yamagishi, N Tanaka, T Oshima

    FEBS LETTERS   410 ( 2-3 ) 141 - 144  1997.06  [Refereed]

     View Summary

    To understand the role of the amino acid residue at position 172 in the conformational stability, four mutant enzymes of Thermus thermophilus 3-isopropylmalate dehydrogenase in which Ala(172) was replaced with Asp, Glu, Asn, and GIn were prepared by site-directed mutagenesis, Three mutants were more stable than the wild-type enzyme, No significant change in catalytic properties was found in the mutant enzymes. The molecular modeling studies suggested that the enhanced thermostability of the mutant enzymes resulted from the formation of extra electrostatic interactions and/or improvement of hydrophobic packing of the interior core. (C) 1997 Federation of European Biochemical Societies.

    DOI PubMed

  • A mutation at the interface between domains causes rearrangement of domains in 3-isopropylmalate dehydrogenase

    CX Qu, S Akanuma, H Moriyama, N Tanaka, T Oshima

    PROTEIN ENGINEERING   10 ( 1 ) 45 - 52  1997.01  [Refereed]

     View Summary

    The structure of a thermostable Ala172Leu mutant, designated A172L, of 3-isopropylmalate dehydrogenase from Thermus thermophilus was determined, The crystal belongs to space group P2(1), with cell parameters a = 55.5 Angstrom, b = 88.1 Angstrom, c = 72.0 Angstrom and beta = 100.9 degrees. There is one dimer in each asymmetric unit, The final R factor is 17.8% with 69 water molecules at 2.35 Angstrom resolution, The mutation is located at the interface between domains and the C alpha trace of the mutant structure deviates from that of the native structure by as much as 1.7 Angstrom, while the structure of each domain barely changes, The mutant enzyme has a more closed conformation compared with the wild-type enzyme as a result of the replacement of Ala with Leu at residue 172, These structural variations were found independent of the crystal packing, because the structure of wild type was the same in crystals obtained in different precipitants. The hinge regions for the movement of domains are located around the active cleft of the enzyme, an observation that implies that the mobility of domains around the hinge is indispensable for the activity of the enzyme, The larger side chain at the mutated site contributed to the thermostability of the mutant protein by enhancing the local packing of side chains, and also by shifting the backbone of the opposing domain.

    DOI PubMed

  • Spontaneous tandem sequence duplications reverse the thermal stability of carboxyl-terminal modified 3-isopropylmalate dehydrogenase

    S Akanuma, A Yamagishi, N Tanaka, T Oshima

    JOURNAL OF BACTERIOLOGY   178 ( 21 ) 6300 - 6304  1996.11  [Refereed]

     View Summary

    A mutant strain of Thermus thermophilus which contains deletions in the 3'-terminal region of its leuB gene showed a temperature-sensitive growth phenotype in the absence of leucine. Three phenotypically thermostable mutants were isolated from the temperature-sensitive strain by spontaneous evolution. Each pseudorevertant carried a tandem sequence duplication in the 3' region of its leuB gene. The mutated 3-isopropylmalate dehydrogenases encoded by the leuB genes from the pseudorevertants were more thermostable than the enzyme from the temperature-sensitive strain. Structural analyses suggested that the decreased thermostability of the enzyme from the temperature-sensitive strain was caused by reducing hydrophobic and electrostatic interactions in the carboxyl-terminal region and that the recovered stability of the enzymes from the pseudorevertants was due to the restoration of the hydrophobic interaction, Our results indicate that tandem sequence duplications are the general genetic way to alter protein characteristics in evolution.

    PubMed

  • Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method

    T Kotsuka, S Akanuma, M Tomuro, A Yamagishi, T Oshima

    JOURNAL OF BACTERIOLOGY   178 ( 3 ) 723 - 727  1996.02  [Refereed]

     View Summary

    We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-172-->Val substitution in all of the mutants. The thermal stability of the thermophile IPMDH was improved by introducing the amino acid substitution.

    PubMed

  • 温度ジャンプラウエ法

    堀 哲哉, 森山 英明, 五十嵐 教之, 赤沼 哲史, 池崎 章, 広瀬 雷太, 大島 泰郎, 田中 信夫

    日本結晶学会誌   38 ( 0 ) 93 - 93  1996

    CiNii

  • IMPROVEMENT OF THERMAL-STABILITY OF ENZYMES IN AN EXTREME THERMOPHILE, THERMUS-THERMOPHILUS - ARTIFICIAL EVOLUTION OF ENZYMES IN THE THERMOPHILE

    A YAMAGISHI, J ABE, M RYOLI, S AKANUMA, M AOSHIMA, T KOTSUKA, M TAMAKOSHI, T OSHIMA

    PROTEIN ENGINEERING   8   3 - 3  1995  [Refereed]

  • THERMOSTABILIZATION OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM AN EXTREME THERMOPHILE, THERMUS-THERMOPHILIS, BY INTRODUCING A BULKY SIDE-CHAIN AT POSITION-172

    S AKANUMA, A YAMAGISHI, T OSHIMA

    PROTEIN ENGINEERING   8   4 - 4  1995  [Refereed]

  • RECOMBINATION-DEFICIENT MUTANTS OF AN EXTREME THERMOPHILE, THERMUS-THERMOPHILUS

    T TAKADA, S AKANUMA, T KOTSUKA, M TAMAKOSHI, A YAMAGISHI, T OSHIMA

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   59 ( 8 ) 2737 - 2739  1993.08  [Refereed]

     View Summary

    Recombination-deficient strains of the extreme thermophile Thermus thermophilus have been prepared from a leucine-isoleucine mutant strain (NM6). The availability of such recombination-deficient thermophilic bacterial strains may provide especially good hosts for work with plasmid vectors.

    PubMed

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Misc

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Industrial Property Rights

  • 低温における酵素活性を向上させた好熱菌由来酵素の改変体の取得方法、及び低温における酵素活性が向上しているサーマス・サーモフィラス由来3-イソプロピルリンゴ酸脱水素酵素の改変体

    赤沼哲史, 木村彦乃, 山岸明彦, 八尾理文

    Patent

  • 白金結合剤、及びその利用

    山岸明彦, 赤沼哲史, 新納寛也, 内田達也, 秋山勇人, 八尾理文

    Patent

  • 単純化されたタンパク質の製造方法

    赤沼哲史, 木川隆則, 横山茂之

    Patent

Awards

  • 第15回酵素応用シンポジウム研究奨励賞

    2014.06   天野エンザイム   進化情報に基づく酵素の耐熱化設計

    Winner: 赤沼 哲史

  • 2011 JB/OUP Poster Prize

    2011.10   Journal of Biochemistry/Oxford University Press  

    Winner: Akanuma Satoshi

  • Alexander von Humboldt Research Fellowship

    2001.03   Alexander von Humboldt Foundation  

    Winner: Akanuma Satoshi

Research Projects

  • 初期翻訳系で使われたアミノ酸種類の時系列変化に関する研究

    JSPS  科研費・基盤研究(B)

    Project Year :

    2017
    -
    2019
     

    赤沼 哲史

  • タンパク質の起源に纏わる「鶏と卵のパラドックス」の解決による地球と宇宙での生命誕生場の推定

    自然科学研究機構・アストロバイオロジーセンター  サテライト研究

    Project Year :

    2018
     
     
     

    赤沼 哲史

  • 冥王代生命学の創成 ・初期地球環境に相対的に多く存在したアミノ酸種を用いた原始的タンパク質の構築

    JSPS  新学術領域研究(研究領域提案型)

    Project Year :

    2017
    -
    2018
     

    赤沼 哲史

  • タンパク質を材料とした革新的繊維素材開発のための技術基盤の構築

    JSPS  科研費・挑戦的萌芽研究

    Project Year :

    2016
    -
    2018
     

    赤沼 哲史

  • 冥王代生命学の創成 ・生命の誕生に必要となるアミノ酸情報量の実験による検証

    JSPS  新学術領域研究(研究領域提案型)

    Project Year :

    2015
    -
    2016
     

    赤沼 哲史

  • 地球型生命におけるアミノ酸情報量の普遍性と特殊性の検証:遺伝暗号とアミノ酸レパートリーの進化

    自然科学研究機構  アストロバイオロジーセンタープロジェクト研究

    Project Year :

    2015
     
     
     

    赤沼 哲史

  • 進化情報に基づく酵素の耐熱化設計

    天野エンザイム  第15回酵素応用シンポジウム研究奨励金

    Project Year :

    2014
     
     
     

    赤沼 哲史

  • Stabilization of industrially useful enzymes by the ancestral design method

    ノボザイムズジャパン  ノボザイムズジャパン研究ファンド

    Project Year :

    2014
     
     
     

    赤沼 哲史

  • 地球型生命におけるアミノ酸情報量の普遍性と特殊性の検証

    自然科学研究機構・新分野創成センター  宇宙における生命研究分野プロジェクト研究

    Project Year :

    2014
     
     
     

    赤沼 哲史

  • 好熱菌酵素を低温高活性化するためのガイドラインの構築

    JSPS  科研費・基盤研究(C)

    Project Year :

    2012
    -
    2014
     

    赤沼 哲史

▼display all

Presentations

  • Protein engineering experiments to explore the evolution of early life

    Akanuma Satoshi  [Invited]

    New Frontier in Protein Design & Engineering 

    Presentation date: 2019.03

  • 11アミノ酸種だけを用いた活性を持つ酵素の再構成

    赤沼 哲史

    第44回生命の起原および進化学会学術講演会 

    Presentation date: 2019.03

  • アストロバイオロジーセンター・サテライト研究報告I

    赤沼 哲史

    アストロバイオロジーセンター・サテライト研究シンポジウム「深海底から宇宙へ、過去から未来へ、分子から社会へ」 

    Presentation date: 2019.01

  • Establishment of mesophilic-like catalytic property on a thermophilic enzyme without loss of thermostability

    Akanuma Satoshi

    The 70th Annual Meeting of the Society of Biotechnology of Japan 

    Presentation date: 2018.09

  • 原始地球上に比較的多く存在したと推定されるアミノ酸種のタンパク質合成における役割

    Akanuma Satoshi

    The 20th Annual Meeting of the Society of Evolutionary Studies, Japan 

    Presentation date: 2018.08

  • Protein engineering studies to explore the early evolution of life

    Akanuma Satoshi

    The 18th Annual Meeting of the Protein Science Society of Japan 

    Presentation date: 2018.06

  • 好熱菌酵素の常温菌酵素域へまでの低温高活性化

    赤沼哲史, シュウギョウトウ, 別所瑞萌, 木村彦乃, 山岸明彦

    私立大学戦略的研究基盤形成支援事業「健康で豊かな生活を実現するスマートタンパク質工学戦略的研究拠点」最終報告会 

    Presentation date: 2018.03

  • 共通祖先

    Akanuma Satoshi  [Invited]

    アストロバイオロジーセンター(ABC)シンポジウム 

    Presentation date: 2018.01

  • 20種未満のアミノ酸によるタンパク質の再構成

    Akanuma Satoshi

    2017年度日本アストロバイオロジーネットワークワークショップ 

    Presentation date: 2017.11

  • 初期地球環境に相対的に多く存在したアミノ酸種を用いた原始的タンパク質の構築

    Akanuma Satoshi

    冥王代生命学の創成・平成29年度 白馬ワークショップ 

    Presentation date: 2017.10

  • Introduction: Dialogue between in vitro evolution and biological evolution, both of which have created new functional biomolecules

    Akanuma Satoshi

    The 55th Annual Meeting of the Biophysical Society of Japan 

    Presentation date: 2017.09

  • RNAワールドを取り巻く未解決課題-核酸合成からRNA-タンパク質ワールドへの移行まで

    Akanuma Satoshi

    19th Annual Meeting of the Society of Evolutionary Studies, Japan 

    Presentation date: 2017.08

  • 初期地球環境に相対的に多く存在したアミノ酸種を用いた原始的タンパク質の構築

    Akanuma Satoshi

    冥王代生命学の創成・平成29年度キックオフワークショップ 

    Presentation date: 2017.05

  • 祖先型酵素改変の進化

    赤沼哲史, 渋江怜, 張博文, 羽室勇飛, 笹本峻弘, 別所瑞萌, 横堀伸一, 山岸明彦

    私立大学戦略的研究基盤形成支援事業「健康で豊かな生活を実現するスマートタンパク質工学戦略的研究拠点」平成28年度年度末報告会 

    Presentation date: 2017.03

  • Thermophilicity of early life inferred by the resurrection of ancient proteins

    Satoshi Akanuma  [Invited]

    Search for life : from early Earth to exoplanets 

    Presentation date: 2016.12

  • Incorporation of evolutionary information into protein design

    Satoshi Akanuma, Shin-ichi Yokobori, Akihiko Yamagishi

    The 39th Annual Meeting of the Molecular Biology Society of Japan 

    Presentation date: 2016.12

  • Creating stable proteins by ancestral design and improving the low-temperature activity of thermophilic ensymes

    Satoshi Akanuma

    私立大学戦略的研究基盤形成支援事業「健康で豊かな生活を実現するスマートタンパク質工学戦略的研究拠点」国際シンポジウム 

    Presentation date: 2016.11

  • Introduction: Advances in the engineering of protein oligomerization and solubility

    Satoshi Akanuma

    The 54th Annual Meeting of the Biophysical Society of Japan 

    Presentation date: 2016.11

  • 学生の頃、何を考え今はやっているか ・ ・ 生命の起源と私

    Akanuma Satoshi  [Invited]

    2016年 生命の起原および進化学会&日本アストロバイオロジーネットワーク 夏の学校 

    Presentation date: 2016.08

  • Experimental evidence for thermophilic ancestral life

    Satoshi Akanuma

    18th Annual Meeting of the Society of Evolutionary Studies, Japan 

    Presentation date: 2016.08

  • Characterization of ancient proteins, life and environment by comparing extant homologous genes

    Satoshi Akanuma, Shin-ichi Yokobori, Takahiro Sasamoto, Mizumo Bessho, Akihiko Yamagishi

    生命の起原および進化学会第41回学術講演会 

    Presentation date: 2016.03

  • Universality and particularity of the amino acid usage of terrestrial life: Evolution of the genetic code and amino acid repertoire

    Akanuma Satoshi

    4th Astrobiology workshop 2016 

    Presentation date: 2016.03

  • 宇宙におけるタンパク質を構成するアミノ酸の種類の可能性

    Akanuma Satoshi

    第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会 

    Presentation date: 2015.12

  • イントロダクション:実用化を見据えた様々な生体関連分子工学の基礎および応用

    Akanuma Satoshi

    第38回日本分子生物学会年会、第88回日本生化学会大会 合同大会 

    Presentation date: 2015.12

  • Reverification of the thermophilicity of early life

    Satoshi Akanuma

    Astrobiology Network Workshop 2015 

    Presentation date: 2015.11

  • 始生代遺伝子の復元と解析による始生代生物と地球環境の推定

    Akanuma Satoshi  [Invited]

    青山学院大学CATプロジェクトシンポジウム 

    Presentation date: 2015.09

  • Stabilization of industrially useful enzymes by the ancestral design method

    Satoshi Akanuma  [Invited]

    Novozymes Japan Biotechnology Forum 2014 

    Presentation date: 2014.10

  • コモノートから遡るアミノ酸の種類

    Akanuma Satoshi

    第16回日本進化学会大会 

    Presentation date: 2014.08

  • 古代タンパク質の復元と解析および超好熱性タンパク質設計への応用

    赤沼哲史, 山岸明彦

    第14回日本蛋白質科学会 

    Presentation date: 2014.06

  • 進化情報に基づく酵素の耐熱化

    Akanuma Satoshi  [Invited]

    第15回酵素応用シンポジウム 

    Presentation date: 2014.06

  • 進化情報に基づく古代タンパク質の復元とタンパク質設計への応用

    Akanuma Satoshi  [Invited]

    第23回 内毒素・LPS研究会 

    Presentation date: 2014.06

  • Experimentally testing the hypothesis of a limited amino acid repertoire in primitive proteins

    Satoshi Akanuma  [Invited]

    International Astrobiology Workshop 2013 

    Presentation date: 2013.11

  • 遺伝暗号の起源

    Akanuma Satoshi  [Invited]

    総研大「惑星科学と生命科学の融合」第3回研究会 

    Presentation date: 2013.08

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Specific Research

  • 祖先復元型リボソーム蛋白質を用いた原始生物の生育環境pHの推定

    2021  

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    原始地球における生命の誕生過程を理解するうえで、生命の誕生を促した原始地球環境を理解することは極めて重要な課題である。これまでの研究では、主に古代生物圏環境の温度に焦点を当ててきたが、本研究では、原始生物の生息環境pHに着目した。既に過去の研究で復元した全生物共通祖先ヌクレオシドニリン酸キナーゼ(NDK)と真正細菌共通祖先リボソームタンパク質S8を用い、それぞれの祖先型タンパク質の酸性、中性、アルカリ性溶液中での熱変性温度を測定した。さらに、現存の好酸性生物、好中性生物、好アルカリ性生物のNDKとS8の熱変性温度のpH依存性と比較することによって、原始生物の生育環境pHを推定した。

  • 共通祖先から遡るタンパク質の起源:原始アミノ酸組成を持つリボソーム蛋⽩質の再構成

    2020  

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    生命の起源に関してはRNAワールド仮説が支持を得ており、次の重要な課題の一つが、現在の生物共通の機能分子であるタンパク質の起源である。生命誕生以前のタンパク質は環境中に存在したアミノ酸から合成されたはずである。原始地球模倣環境での放電実験や隕石の分析から、原始地球には、現在のタンパク質合成に使われる20種類のアミノ酸の約半数が存在した可能性が指摘された。本研究では、リボソームRNAとの結合活性を保持したリボソーム蛋白質を、原始地球に比較的豊富に存在したと推定された少数種類のアミノ酸から再構成可能か実験による検証をおこなった。

  • プレバイオティックアミノ酸によるRNA結合タンパク質の再構成

    2020  

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    生命の起源に関しては多くの証拠からRNAワールド仮説が有力視されている。一方、現存生物が共通して持つ機能生体高分子であるタンパク質がRNAワールドにどのように誕生し、RNAワールドがRNA-タンパク質ワールドにどのように拡張したかが次の課題である。本研究では、現存のすべての生物が持つ、RNAとタンパク質の複合体であり、タンパク質合成の場でもあるリボソームに着目した。共通祖先生物が持っていたと推定された祖先型リボソームタンパク質S7とS8を復元し、祖先型リボソームタンパク質の安定性解析と機能解析を実施した。

  • 原始アミノ酸だけによるRNA結合タンパク質の構築

    2019   古川龍太郎, 趙方正

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    生命の起源の解明に向けてRNAワールド仮説が支持を広げているが、次の課題となるのがRNAワールドにどのようにタンパク質が誕生したかである。本研究では、RNA結合タンパク質の代表例として、RNA機能に対して補助的ではあるが必須の役割を担っているリボソームタンパク質S7とS8を研究材料として選択し、原始地球環境に比較的豊富に存在したと推定されるアミノ酸種だけからS7とS8を再構成する試みを進めている。現在までに、進化系統解析と人工遺伝子合成により共通祖先型S7とS8を復元した。さらに、祖先型S7とS8から特定のアミノ酸種を欠損させたアミノ酸組成単純化型改変体の再構成もおこなっている。

  • 祖先タンパク質再構成による初期地球生物圏の環境pHの推定

    2018  

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    地球上で最初の生命が誕生した前後の初期地球における生物生息圏の環境pHついては、いくつかの議論があるものの、当時の地質学的情報が少ないため、未だに明確な知見は得られていない。本研究では、進化系統解析と遺伝子工学的手法によって復元した、全生物の共通祖先が持っていたと推測されるタンパク質の安定性のpH依存性を測定した。復元した全生物の共通祖先タンパク質は中性で最も安定であり、酸性とアルカリ性では安定性が大きく低下した。同様の傾向が、中性環境に生息する現存生物の複数のタンパク質でも見られた。以上の結果から、全生物の共通祖先は中性環境に生息していたと推定された。

  • 耐熱性と低温活性を両立した酵素の創成

    2017  

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    好熱菌酵素は優れた耐熱性を持つが、低温では活性が大幅に低下する。これまでの研究では、高度好熱菌Thermus thermophilus由来3-イソプロピルリンゴ酸脱水素酵素(IPMDH)のアミノ酸配列と、大腸菌由来IPMDHのアミノ酸配列を比較し、両者でアミノ酸が異なる部位に、常温菌IPMDHに見られるアミノ酸を好熱菌IPMDHにアミノ酸置換により導入した変異型酵素を作製した。今年度は、すでに作製された変異型酵素の詳細な触媒活性の解析をおこなった。その結果、わずか3アミノ酸置換によって、好熱菌酵素の高い耐熱性を損ねることなく、低温活性を常温菌酵素と同程度にまで改善できることを明らかにした。

  • 進化情報を利用した担子菌由来ポリフェノール重合化酵素の安定化改変

    2016  

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    Laccase is a kind of polyphenol oxidases and frequentlyfound among higher plants and fungi. We previously cloned a laccase-coding genefrom a cDNA library of the Royal Sun medicinal mushroom, Agaricus brasiliensis. However, our efforts to yield the wild-typelaccase was unsuccessful probably due to its instability. Therefore, in thisstudy, we tried to improve the stability of the laccase by the ancestral designmethod. The designed laccase was successfully yielded by a Pichia pastoris secretoryexpression system and then purified. Thermal stability measurements of thepurified laccase showed that the protein is nicely thermostable; its half-lifeat 70ºC is almost 70 min. The half-inactivationtemperature of the protein after heat-treatment for 10 min is higher than 80ºC. The kinetic properties are reasonably similarto those of a laccase from a closely related species, A. bisporus. We also found that the designed laccase can catalyze the polymerization of severalmonomeric polyphenols.

  • 好熱菌酵素の効率的低温高活性化法の開発

    2015  

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    好熱菌酵素の工業利用の推進には、高い安定性を維持したまま、低温活性を向上させるための改変法の開発が課題である。この目的のため、好熱菌と大腸菌酵素のアミノ酸配列を比較し、両者で異なるアミノ酸のうち、活性部位に近いアミノ酸を大腸菌型に置換することによって、効率的に好熱菌酵素の低温活性を改善できるか検証した。計29変異体を作製し、それらの25℃における比活性を測定したところ、10変異体が好熱菌野生型酵素と比べて2倍以上大きい活性を示した。さらに、有益なアミノ酸置換を組み合わせた結果、最大で野生型酵素の14倍の比活性を持つ変異体が得られた。加えて、この変異体の変性温度は85℃と高い耐熱性を維持していた。

  • 進化情報に基づく好熱菌酵素の効率的低温高活性化法の確立

    2015  

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    様々な条件で安定な好熱菌酵素は多くの工業用途が期待されているが、低温で触媒活性が著しく低下することが利用の妨げとなっている。そこで、相同アミノ酸配列の比較に基づく好熱菌酵素の低温高活性化法の確立に取り組んできた。本研究では、好熱菌酵素と常温菌相同酵素とでアミノ酸が異なる部位のうち、活性部位に近い範囲に低温高活性化に関わるアミノ酸が多くある可能性と、耐熱性の犠牲無しに低温活性を改善できる可能性を検証した。その結果、活性部位からの距離と低温活性化に寄与するアミノ酸置換が見つかる頻度には相関がないことと、耐熱性をほとんど低下させることなく25℃の比活性を10倍以上向上できることを明らかにした。

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Syllabus

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